A new and innovative electron paramagnetic resonance (EPR) methodology will be developed for in vivo spectroscopy, imaging and biomedical research. A partnership of engineers, research scientists, clinicians, and industry propose to design and build rapid-scan EPR systems operating at 250 MHz and at L-band (ca. 1.1 GHz). The emphasis is on careful engineering of the spectrometer systems, including magnet, scan coils, resonator, and data acquisition to optimize physiological EPR signal acquisition for in vivo spectroscopy and imaging. Rapid-scan EPR encompasses the regime in which the magnetic field sweep is fast relative to relaxation times, which is a newly developed intermediate regime between CW and pulsed EPR. Direct-detection rapid-scan EPR signals provide the absorption lineshape directly, reveal electron spin relaxation times without requiring high incident power, and provide accurate relative amplitudes of peaks in rapidly decaying signals. The Specific Aims are: (1) Build a dedicated rapid-scan spectrometer at L-band (ca. 1.1 GHz) with scan rates optimized for biomedical, in vivo, and imaging experiments. (2) Build improved resonators, rapid scan coils and drivers, and dedicated rapid-scan bridge at 250 MHz. (3) Build a 250 MHz rapid scan bridge, resonator, and magnetic field scan coil unit at the University of Denver and install it at the University of Chicago Center for In Vivo Imaging of Physiology, where it will be used to image oxygen concentrations in animal tumors. The benefits of traditional CW imaging, pulsed EPR imaging, and rapid-scan imaging for oximetry and oncology will be compared. (4) Design, build and test hardware/software systems for acquisition of the rapid-scan signal and post-processing of spectral information that will be used at 250 MHz and at L-band. In addition to in vivo imaging, future applications include the study of transient paramagnetic species, such as spin-trapped radicals with short lifetimes, time-dependent biological processes, and fundamental spin relaxation phenomena. This instrumentation is a new enabling technology and will create a new era for measuring oxygen in vivo and for scientific investigation of paramagnetic species, including in vivo free radicals. Free radicals are implicated in many diseases, and measurement of oxygen in vivo is important for cancer treatment, peripheral vascular disease, and wound healing.